PROJECT SUMMARY Protein translocation across lipid bilayers is a fundamental step in many biological processes, such as protein sorting and secretion in eukaryotic cells and delivery of cytotoxic components by pathogenic bacteria. Membrane-spanning proteins that translocate proteins and peptides are known as ?translocase channels.? The importance of the interaction between translocase channels and the membrane lipids that surround them as well as the molecular basis of protein translocation are poorly understood. The activity of many transmembrane proteins and protein complexes, including translocase channels, can depend on the presence of specific types of lipid in the membranes where they insert. Characterizing lipid recruitment to the vicinity of the channels holds promise for developing therapeutic drugs targeted at lipid-protein interactions, but studying the precise nanoscale lipid environment of membrane proteins can be exceptionally challenging with current methods. Likewise, elucidating the mechanisms by which translocase channels transport proteins, which may be much larger than the channel's pore, across membranes is of fundamental and therapeutic interest. Anthrax toxin, secreted by Bacillus anthracis, is a model translocation system that exhibits host cell lipid dependencies and functions by transporting ~90 kDa cytotoxic proteins through a pore that is only ~6-12 in diameter. The toxin's three components are known as protective antigen (PA), lethal factor (LF), and edema factor (EF). These toxins co- assemble on the host cell surface after binding to host cell receptor proteins and are then endocytosed via a cholesterol-dependent pathway. Acidification of the endosome causes the PA proteins to form a narrow aqueous channel across the endosomal membrane, and the enzymes LF and EF unfold, pass through the channel, and refold inside the cytosol, where they carry out their cytotoxic roles. It is unknown to what extent the channel recruits specific classes of lipids or how lipid-protein interactions affect its structure and function. Current models for LF translocation suggest that it proceeds via a Brownian ratchet-like or helix compaction mechanism, but many questions remain unanswered as to the crucial initial step of inserting unfolded LF or EF into the long, narrow pore. The lipid stoichiometry of lipid-protein Nanodiscs containing with anthrax lethal toxin and varied lipid composition will be determined using native ion mobility-mass spectrometry (IM-MS). After pH-induced insertion of LF into the PA pore within the Nanodisc environment, native IM-MS will be used to characterize changes in the size and shape of the intact complex. The preferred lipid environment of the pore and the intrinsic entropic barrier to LF insertion through the pore will thereby be elucidated. It is expected that the pore preferentially recruits raft-associated lipids and that LF translocation occurs via a large number of energetically competitive intermediate states. Relevance: Characterizing nanoscale lipid recruitment and entropic factors in the initial stages of translocation by anthrax toxin is relevant to development of targeted methods to inactivate the toxin and novel technologies that exploit translocase channels as sensors or drug-delivery tools.